Gram stain

Last revised by Daniel J Bell on 11 May 2020

The Gram stain (or Gram method) is a key microbiological method for staining bacteria. The process relies upon two stains, the first, a complex of crystal violet and iodine, and the second, safranin, a red counterstain. The staining procedure subdivides bacteria into Gram-positive and Gram-negative bacteria, with important implications for their further categorization and treatment 1,2

It is a straightforward rapid staining method detailed in the reference. After each treatment step the slide is washed with water for 5 seconds to remove any excess reagent and excess water shaken off 2:

  1. bacterial smear is heat-fixed to the glass slide
  2. smear is initially covered with crystal violet for 30 seconds
  3. slide washed
  4. smear is treated with Gram's iodine solution for one minute
  5. slide washed
  6. decolourisation with 1:1 95% ethanol-acetone solution
  7. slide washed
  8. covered with counterstain, usually safranin O (in ethanol)
  9. slide washed
  10. smear is viewed with high-magnification oil immersion microscopy to decide if Gram positive or negative
  • Gram-positive bacteria are purple
  • Gram-negative bacteria are pink

Fundamentally the method depends upon the difference in cell wall composition of the two types of bacteria. The cell walls of Gram-positive organisms are thick and retain the violet-iodine complex after treatment with alcohol retaining a purple hue. By contradistinction, in gram-negative organisms the cell wall (as it is) is much thinner and the initial dye is washed out by the decolourant, and the micro-organisms retain only the counterstain, rendering them pink (the counterstain attaches to the Gram-positive bacteria but cannot be seen due to being overwhelmed by the dark purple color of the crystal violet.

The Gram stain was developed by a Danish physician, Hans Christian Joachim Gram (1853-1938), whilst he studied methods to double stain renal histopathological specimens in Copenhagen in 1883. Serendipitously he found that some bacteria remained color-fast after being washed with alcohol 3

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